CN114487408B - Application of Arginase-2 as a target in screening products for diagnosis, prevention and/or treatment of drug-induced renal injury - Google Patents
Application of Arginase-2 as a target in screening products for diagnosis, prevention and/or treatment of drug-induced renal injury Download PDFInfo
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Classifications
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
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- G01N2333/978—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/34—Genitourinary disorders
- G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
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- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Urology & Nephrology (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract
本发明提供一种Arginase‑2作为靶点在筛选诊断、预防和/或治疗药源性肾损伤的制品中的应用，发明人发现在发生了药源性肾损伤的人体和动物标本中，对比剂或顺铂等肾毒性药物可导致血液中的Arginase‑2水平显著升高，动物肾脏中的Arginase‑2蛋白表达明显上调；同时，药物抑制或基因沉默Arginase‑2的表达，可明显增加药源性肾损伤小鼠的尿量，降低血肌酐水平，缓解对比剂等导致的肾小管上皮细胞硝化应激，下调HO‑1蛋白表达，显著逆转对比剂等导致的肾小管上皮细胞凋亡和铁死亡，由此将Arginase‑2作为干预靶标用于筛选诊断、预防和/或治药源性肾损伤的制品。
The present invention provides an application of Arginase-2 as a target in screening products for diagnosis, prevention and/or treatment of drug-induced renal injury. The inventors found that in human and animal specimens with drug-induced renal injury, nephrotoxic drugs such as contrast agents or cisplatin can cause a significant increase in the level of Arginase-2 in the blood, and the expression of Arginase-2 protein in the kidneys of animals is significantly upregulated; at the same time, drug inhibition or gene silencing of Arginase-2 expression can significantly increase the urine volume of mice with drug-induced renal injury, reduce blood creatinine levels, alleviate the nitrative stress of renal tubular epithelial cells caused by contrast agents, etc., downregulate HO-1 protein expression, and significantly reverse the apoptosis and ferroptosis of renal tubular epithelial cells caused by contrast agents, etc., thereby using Arginase-2 as an intervention target for screening products for diagnosis, prevention and/or treatment of drug-induced renal injury.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to an application of Arginase-2 serving as a target in screening, diagnosing, preventing and/or treating medicine-induced kidney injury products.
Background
Acute kidney injury (acutekidneyinjury, AKI) is a major global health problem associated with high morbidity, increased medical costs, and high mortality. Furthermore, while AKI is considered a reversible syndrome, it often leads to Chronic Kidney Disease (CKD). Drug-induced kidney injury is a major cause of AKI, including contrast agent use, cisplatin drug treatment, and the like. Iodine contrast agent (also called iodine contrast agent hereinafter referred to as contrast agent) is a kind of medicine widely used in radio operation for enhancing development effect, improving diagnosis and treatment level, and the annual usage amount of the iodine contrast agent exceeds 7500 ten thousand per year, and is mainly used for angiocardiography, urography, other official cavity contrast agent examination, computer tomography (Computedtomography, CT) enhancement and the like. However, 11% -50% of patients may develop contrast nephropathy (calculated-induced acute kidney injury, CI-AKI) after administration of contrast agent, i.e., contrast-induced acute kidney injury, which is defined as an increase in blood creatinine levels of 25% or 44.2 μmol/L compared to baseline within 72 hours after intravascular administration of contrast agent without the influence of factors such as surgery, nephrotoxic drugs, etc. Clinical researches show that CI-AKI accounts for about 12% of iatrogenic acute kidney injury, the death rate caused by the CI-AKI is increased by 4-5 times, the rate of chronic renal failure and total death rate is as high as 7-31%, the average hospitalization time and socioeconomic burden are increased by 5-10 times, the CI-AKI becomes an important cause affecting the health of residents in China, and the economic burden of diseases is increased gradually. However, the kidney injury caused by the contrast agent still lacks effective prevention measures, the prevention measures of CI-AKI such as hydration treatment, antioxidation treatment and the like are all nonspecific in clinic, and the occurrence rate of CI-AKI cannot be reduced through the discovery of the latest large-scale clinical research, so that the finding of a specific target point for developing a new medicament for diagnosing, preventing and/or treating the drug-induced kidney injury such as the contrast agent nephropathy is a technical problem to be solved urgently.
Disclosure of Invention
Arginase is an enzyme responsible for the urea cycle and hydrolyzes L-arginine (L-arginine) to ornithine and urea. Arginase has two subtypes, arginase 1 (Arginase-1) is mainly expressed in the liver, and arginase 2 (Arginase-2) is mainly expressed in the kidney, and particularly has high expression in the extramedullary streaks of the kidney. Arginase-2 regulates NO production by competing with Nitric Oxide (NO) Synthase (NOs) for the same substrate l-arginine. The inventor finds that in human and animal specimens with drug-induced kidney injury, contrast agents or nephrotoxic drugs such as cisplatin and the like can cause Arginase-2 level in blood to be obviously increased, and Arginase-2 protein expression in animal kidneys is obviously up-regulated. The increased level of Arginase-2 in the kidney tissue increases the consumption of L-arginine and results in a decrease in the availability of NOS, thereby inducing the uncoupling of NOS, mediating nitrosation stress and ultimately damaging the kidney tissue.
Based on the above, the invention provides a therapeutic target with potential therapeutic effects on drug-induced kidney injury such as contrast nephropathy, and can relieve renal tubular epithelial cell nitrification stress caused by contrast agent and the like, down regulate HO-1 protein expression, obviously reverse renal tubular epithelial cell apoptosis and iron death caused by contrast agent and the like, improve urine volume of mice and reduce creatinine level of the mice through nonspecific drug inhibition (nor-NOHA) or gene intervention silencing Arginase-2 expression. In particular, the primary object of the present invention is the use of Arginase-2 as a target in the screening of a preparation for the treatment of drug-induced kidney injury.
It is a second object of the present invention to provide the use of Arginase-2 as a target in screening for a preparation for preventing a drug-induced kidney injury.
It is a further object of the present invention to provide the use of Arginase-2 as a target in screening for a product for alleviating a drug-induced kidney injury.
Further, in any of the above aspects, the article of manufacture includes, but is not limited to, a pharmaceutical or biochemical agent. When the product is a medicament, the medicament can contain one or more pharmaceutically acceptable carriers, and the medicament can be further prepared into corresponding medicament preparations by a conventional method in the pharmaceutical field, and the medicament can also contain one or more other components with the same or similar activity as the invention or components with different activities from the invention, wherein the components can enhance the activities in a plurality of schemes of the invention, or in some cases, auxiliary materials or other active components can reduce the activities in any of the schemes of the invention.
Further, in any of the above embodiments, the drug-induced kidney injury is a kidney injury caused by a contrast agent and/or a nephrotoxic drug. In particular, the contrast agent includes but is not limited to iohexol and the like, and the nephrotoxic drug includes but is not limited to cisplatin and the like.
The fourth object of the invention is to provide the application of Arginase-2 as a target in screening products for diagnosing drug-induced kidney injury.
In particular, the articles include, but are not limited to, pharmaceuticals or biochemical agents. When the product is a medicament, the medicament can contain one or more pharmaceutically acceptable carriers, and the medicament can be further prepared into corresponding medicament preparations by a conventional method in the pharmaceutical field, and the medicament can also contain one or more other components with the same or similar activity as the invention or components with different activities from the invention, wherein the components can enhance the activities in a plurality of schemes of the invention, or in some cases, auxiliary materials or other active components can reduce the activities in any of the schemes of the invention. More specifically, the article of manufacture includes, but is not limited to, a kit.
It is a fifth object of the present invention to provide the use of Arginase-2 as a target in screening for an agent for down-regulating the expression level of HO-1 or Arginase-2 protein.
Specifically, the preparation can take Arginase-2 as a target to carry out gene mutation or gene silencing, or the preparation is a substance for inhibiting activity or expression of Arginase-2. The article of manufacture may be a pharmaceutical or biochemical agent.
It is a sixth object of the present invention to provide the use of Arginase-2 inhibitors or of preparations containing Arginase-2 inhibitors for the prophylaxis of kidney injury of medical origin.
The seventh object of the present invention is to provide the use of Arginase-2 inhibitors or of preparations containing Arginase-2 inhibitors for the treatment of kidney injuries of medical origin.
The invention also provides application of Arginase-2 inhibitor in preparation of a product for treating drug-induced kidney injury.
The invention provides a use of Arginase-2 inhibitor in preparation of a preparation for preventing drug-induced kidney injury.
In particular, the Arginase-2 inhibitors include, but are not limited to, nor-NOHA.
In some preferred embodiments, in any of the above embodiments, the drug-induced kidney injury is a kidney injury caused by a contrast agent and/or a nephrotoxic drug, and the article of manufacture includes, but is not limited to, a drug or a biochemical agent. When the product is a medicament, the medicament can contain one or more pharmaceutically acceptable carriers, and the medicament can be further prepared into corresponding medicament preparations by a conventional method in the pharmaceutical field, and the medicament can also contain one or more other components with the same or similar activity as the invention or components with different activities from the invention, wherein the components can enhance the activities in a plurality of schemes of the invention, or in some cases, auxiliary materials or other active components can reduce the activities in any of the schemes of the invention.
The application of any of the above embodiments does not include diagnosis and treatment of diseases.
Compared with the prior art, the invention has the following beneficial effects:
The invention discovers a target spot-Arginase-2 with potential diagnosis, prevention and/or treatment of drug-induced kidney injury, and provides a new idea for realizing diagnosis, prevention and/or treatment of drug-induced kidney injury by drug inhibition or gene silencing Arginase-2 expression, relieving renal tubular epithelial tissue nitrifying stress caused by contrast agents, renal toxicity drugs and the like, down-regulating HO-1 protein expression, obviously reversing apoptosis and iron death of renal tubular epithelial cells caused by contrast agents, renal toxicity drugs and the like, and provides a product for screening diagnosis, prevention and/or treatment of drug-induced kidney injury by taking Arginase-2 as a target spot.
Drawings
FIG. 1 is a diagram showing the detection of Arginase-2 protein expression levels in contrast-treated HK-2 cells by Westernblot;
FIG. 2 shows the morphology change of the renal tubular epithelial cells caused by contrast media under electron microscopy, wherein panels A and B are electron microscopy images of HK-2 cells treated without iohexol in example 1, panels C and D are electron microscopy images of HK-2 cells treated with iohexol in example 1, panels E and F are morphology change images of renal tubular epithelial cells of rats without iohexol intervention (tail vein injection iohexol) in example 2, and panels G and H are morphology change images of renal tubular epithelial cells of rats with iohexol intervention (tail vein injection iohexol) in example 2;
FIG. 3 is a model of a Arginase-2 expressing knockdown HK-2 cell;
FIG. 4 is a graph showing that Arginase-2 gene silencing significantly reverses the reduction in NO production by iohexol-induced HK-2 cells;
FIG. 5 is a graph showing that Arginase-2 gene silencing significantly reverses the increased 3-NT production by iohexol in HK-2 cells;
FIG. 6 shows that iohexol can result in reduced HO-1mRNA and protein expression in HK-2 cells, and Arginase-2 gene silencing can significantly up-regulate HO-1 protein expression levels;
FIG. 7 is a graph showing that Arginase-2 gene silencing significantly reverses the reduction in ATP production and decreased cell viability of HK-2 cells caused by iohexol;
FIG. 8 is a graph showing that Arginase-2 inhibitors significantly increased urine volume and decreased blood creatinine levels in CI-AKI mice;
FIG. 9 shows that the serum Arginase-2 levels in CI-AKI mice and clinical patients were significantly higher than in the control group.
Detailed Description
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. The invention may be embodied in many other forms than described herein and similarly modified by those skilled in the art without departing from the spirit or scope of the invention, which is therefore not limited to the specific embodiments disclosed below.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
In the following examples, unless otherwise indicated, all procedures used are conventional in the art and all reagents used may be prepared by commercial routes or by known published methods.
The invention is completed by three ways of cells, animals and clinical samples.
Example 1 cell pathways
Normal HK-2 cells and genetically silenced HK-2 cells were treated using a contrast agent-induced tubular epithelial cell injury modeling method. The MTT method is used for measuring the cell activity, the morphological change of the tubular epithelial cells is observed by a transmission electron microscope, the changes of mRNA and protein expression levels of Arginase-2 and HO-1 are detected by Real-time PCR and Westernblot, the nitrification stress indexes such as NO, 3-NT, NOS monomers/dimers and the like and apoptosis related indexes are detected, and the ATP level is measured. The specific implementation mode is as follows:
1. A comparison reagent-induced HK-2 cell injury model is established, according to the earlier working basis of the inventor, the concentration of iohexol is determined to be 75mg (I)/mL, the treatment time is 4h, the treatment results are shown in fig. 1 and 2, the treatment result is shown in fig. 1, the treatment of iohexol for 4h can lead to the increase of the expression level of HK-2 cell Arginase-2 protein, in fig. 2, A, B is shown in the drawing of HK-2 cell without iohexol, C, D is shown in the drawing of HK-2 cell incubated with iohexol, in the drawing of A, B in fig. 2, the cell structure is complete, cell membranes, cell nuclei, mitochondria and other organelles are normal, in the drawing of C, D, the organelles are obviously swollen, the mitochondrial membrane is broken, the crest is reduced or vanished, the endoplasmic reticulum is obviously swollen, and the endoplasmic reticulum fracture density is partially increased.
2. A Arginase-2 gene-silenced HK-2 cell line was established by transfecting HK-2 cells with a lentiviral shRNA-Arginase-2 vector, a Arginase-2 gene-silenced HK-2 cell line was established, arginase-2 was detected by Real-time PCR and Westernblot, and the transfection effect was confirmed, and the results of the treatment are shown in FIG. 3. As shown in FIG. 3, the inhibition effect of siRNA3 on Arginase-2 expression is most obvious, the expression rate is about 10%, and the Arginase-2SiRNA3 target sequence is GAGGGCATATTGTCTATGA. The specific operation is as follows:
1) 1X 10 6～5×106 HK-2 cells were seeded in 6-well plate culture wells containing 1863. Mu.L of complete medium;
2) Diluting siRNA by diluting 5. Mu.L of 50nM siRNA stock solution with 120. Mu.L of 1X riboFECTTM CP Buffer, gently shaking;
3) Preparing a mixed solution, namely adding 3 mu L riboFECTTM CP REAGENT, lightly blowing and uniformly mixing, and incubating for 0-15 min at room temperature to prepare a transfection compound;
4) Adding riboFECTTM CP transfection complex into a proper amount of non-double antibody complete culture medium, and gently mixing;
5) Placing the culture plate in a CO 2 incubator at 37 ℃ for culturing for 24-96 hours;
siRNA was transfected into 6 well plates at riboFECTTM CP REAGENT nM.
3. Quantitative detection of NO concentration 1) detection of intracellular NO concentration by DAF-FMDA fluorescent probe according to the method of NO kit (Biyun-NO fluorescent probe quantitative detection kit), diluting DAF-FMDA with DAF-FMDA diluent provided by the kit according to the ratio of 1:1000 to make its final concentration 5. Mu. Mol/L, removing cell culture solution in six well plates, adding 1mL of diluted DAF-FMDA, incubating in a 37 ℃ cell incubator for 20min, washing cells 3 times with PBS (pH 7.4) to sufficiently remove DAF-FMDA which does not enter cells, detecting and analyzing by using an Operetta high content imaging system (Perkinelmer), using excitation wavelength 495, and emission wavelength 515nm. FIG. 4 shows that Arginase-2 gene silencing significantly reversed the reduction in NO production by iohexol-induced HK-2 cells.
4. Real-timePCR and Westernblot analysis, namely detecting the transcription of HO-1mRNA gene and the change of the expression levels of 3-NT and HO-1 proteins, and the analysis results are shown in FIG. 5 and FIG. 6. FIG. 5 shows that Arginase-2 gene silencing can significantly reverse the increased 3-NT production by iohexol in HK-2 cells, FIG. 6 shows that contrast agents can lead to decreased HO-1mRNA and protein expression in HK-2 cells, and Arginase-2 gene silencing can significantly up-regulate HO-1 protein expression levels.
5. Cell proliferation assay (MTT) to measure cell viability, kit to measure cell ATP level, planting 5000 cells/well in 96-well plate, adding MTT solution 20 μl and 180 μl blank medium into each well, incubating in cell incubator for 4 hr, adding 150 μl LFormazan solution into each well, mixing well, measuring absorbance at 490nm with enzyme-labeled instrument to measure cell viability, and measuring ATP level according to ATP kit operation method, and the test results are shown in FIG. 7. FIG. 7 shows that Arginase-2 gene silencing significantly reversed iohexol-induced decreases in ATP production and decreased cell viability in HK-2 cells.
EXAMPLE 2 animal pathway
Male C57BL/6N mice (6-8 weeks) were grouped into control, CI-AKI model, CI-AKI model+nor-NOHA (Arginase-2 inhibitor). Collecting urine in 24h through a metabolism cage, measuring and recording urine volume, taking orbital blood after molding for detecting kidney function evaluation indexes such as blood creatinine and urea nitrogen level, evaluating morphology structure and damage condition of kidney tissues and kidney tubules through HE staining and kidney tubule damage score, observing morphological changes of kidney tubule epithelial cells through a transmission electron microscope, detecting mRNA and protein expression level changes of Arginase-2 and HO-1 in the kidney tissues and the kidney tubules, and detecting nitrification stress indexes such as NO, 3-NT, NOS monomer/dimer and the like and apoptosis related indexes. The specific implementation mode is as follows:
(1) Control, CI-AKI mouse model, CI-AKI model+nor-NOHA (Arginase-2 inhibitor) group were prepared:
Male C57BL/6N mice (6-8 weeks), were anesthetized with 0.6% sodium pentobarbital (80 mg/kg) and right nephrectomized, dehydrated for 48h 3 weeks after surgery. The control mice were intraperitoneally injected with 100mg/kg of nor-NOHA (Arginase-2 inhibitor) after 48h of dehydration, 10mL/kg of furosemide after 30min of dehydration, 15mL/kg of 0.9% physiological saline was intraperitoneally injected with a 30min interval, the CI-AKI mice model group was intraperitoneally injected with 100mL/kg of 0.9% physiological saline after 48h of dehydration, 10mL/kg of furosemide after 30min of dehydration, 15mL/kg of iohexol (350 mg (I)/mL) was intraperitoneally injected with a 30min interval, and the CI-AKI models group + nor-NOHA (Arginase-2 inhibitor) groups were intraperitoneally injected with 100mg/kg of nor-NOHA (Arginase-2 inhibitor) after 48h of dehydration, 10mL/kg of furosemide was intraperitoneally injected with a 30min interval, and 15mL/kg of iohexol (350 mg (I)/mL) was intraperitoneally injected with a 30min interval. Finally, the mice are singly placed in a metabolism cage for free diet, urine is collected for 24 hours, and the urine volume is measured and recorded. Blood was collected from the orbit 24h later, blood creatinine and urea nitrogen levels were detected, kidney tissue was isolated, tissue sections were 3 μm thick, HE staining and tubular injury scoring were performed to evaluate the morphology and injury of kidney tissue and tubular, and changes in the structure of proximal tubular cells were observed by transmission electron microscopy (magnification, ×5000), as shown in FIG. 8. The results in FIG. 8 show that Arginase-2 inhibitors significantly increased urine and decreased serum creatinine levels in CI-AKI mice, and FIG. 2 (E, F, G, H) shows that rats were free of (E, F) or were treated with (G, H) iohexol (15 mL/kg of iohexol for 24H) and that the control (E, F) had intact cell structures, normal cellular membrane, nuclear and mitochondrial organelles (×2000, ×5000), and that iohexol (G, H) organelles were significantly swollen, mitochondrial membrane damage, reduced or absent, significant swelling of the endoplasmic reticulum, and increased fractional endoplasmic reticulum break densities. As shown in Panel A of FIG. 9, CI-AKI mice have significantly elevated serum Arginase-2 levels compared to the control group.
Example 3 clinical specimens
Venous blood of clinical patients before and after radiography for 6h, 12h, 24h, 48h and 72h is collected, placed in EDTA tube, centrifuged for 10min at 3000r/min, and the supernatant is taken and stored in a refrigerator at-80 ℃. The biochemical automatic analyzer detects the levels of creatinine, urea nitrogen and cystatin C, ELISA method is used to detect Arginase-2 levels, and the result is shown in the diagram B in figure 9. Panel B in FIG. 9 shows that CI-AKI patient (n=6) serum Arginase-2 levels are significantly higher than non-CI-AKI patient (n=6), and that control group (n=3) patients not using contrast agent but matched clinical features have the lowest serum Arginase-2 levels.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (6)
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